Genotoxic mechanisms of asbestos fibers: role of extranuclear targets

Our plain-English summary of Genotoxic mechanisms of asbestos fibers: role of extranuclear targets, by A. Xu, X. Huang, Y.C. Lien, et al (Chemical Research in Toxicology, 2007).

The study

• Authors: A. Xu, X. Huang, Y.C. Lien, L. Bao, Z. Yu, T.K. Hei
• Journal: Chemical Research in Toxicology
• Published: 2007;20(5):724-33
• Cited by: 12 articles, as counted by NIH iCite
• Read it: PubMed · Publisher

Abstract

Asbestos fibers are carcinogenic to both humans and experimental animals. The continued discoveries of exposure routes whereby the general public is exposed to asbestos suggest a long-term, low-dose exposure for a large number of people. However, the mechanisms by which asbestos induces malignancy are not entirely understood. In previous studies, we have shown that asbestos is an effective gene and chromosomal mutagen when assayed using the highly sensitive AL mutation assay and that the mutagenicity is mediated by reactive oxygen species. The objective of the present study is to determine the origin of these radical species, particularly reactive nitrogen species, in fiber mutagenesis. Using the radical probe 5',6'-chloromethyl-2',7'-dihydroxyphenoxazine diacetate to trap reactive radical species, we showed that crocidolite increased the levels of oxyradicals in cytoplasts, in the absence of the nucleus, in a dose-dependent manner, which was reduced significantly by cotreatment with the radical scavenger dimethyl sulfoxide. Treatment of enucleated cells with crocidolite asbestos followed by rescue fusion using karyoplasts from control cells resulted in significant mutant induction, indicating that the nuclear-cytoplasmic interaction is necessary for fiber mutagenesis. Using the fluorescent probe 2,3-diaminonaphthotriazole, crocidolite fibers were shown to induce a dose-dependent increase of nitric oxide production, which was suppressed significantly by concurrent treatment with the nitric oxide synthase inhibitor, NG-methyl-L-arginine (L-NMMA). Similarly, there was a dose-dependent decrease in the mutation yield induced by crocidolites in the presence of graded doses of L-NMMA. These data showed that extranuclear targets play an essential role in the initiation of oxidative damage that mediates fiber mutagenesis in mammalian cells.

In plain English

This laboratory experiment exposed human-hamster hybrid (AL) cells in culture to standard reference crocidolite asbestos fibers. To test whether the part of the cell outside the nucleus could start mutation-causing damage, the authors removed the nucleus from some cells. In these nucleus-free cells, a 6 µg/cm2 dose of crocidolite raised a fluorescent signal for oxygen radicals more than four-fold above controls. Adding the radical scavenger DMSO reduced that signal by more than two-fold. When fiber-treated nucleus-free cells were fused with nuclei from untreated cells, the mutation yield was more than two-fold that of control fused cells. In whole cells, the nitric oxide blocker L-NMMA (15 µM) cut crocidolite-induced mutations by more than three-fold and cut nitric oxide formation by 5-6-fold. An inactive form of the blocker had essentially no effect on mutations. The authors concluded that targets outside the nucleus play an essential role in starting the oxidative damage behind fiber-induced mutations. They noted that treated cells likely still held fiber remnants after washing.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Laboratory (in vitro) cell culture experiment using cells with the nucleus removed, followed by cell fusion techniques
• Who was studied: Cultured human-hamster hybrid (AL) cells, which contain a standard set of CHO-K1 hamster chromosomes and a single copy of human chromosome 11. Some were whole cells, some had the nucleus removed (cytoplasts), and some were cytoplasts fused with nuclei (karyoplasts) from untreated cells. No people were studied.
• Exposure: International Union Against Cancer (UICC) standard reference crocidolite fibers at graded doses (2, 4 and 6 µg/cm2 in the main experiments). Some cells were also treated with DMSO (0.5%), with the nitric oxide synthase inhibitor L-NMMA, or with its inactive form D-NMMA.
• Main results:
  • In nucleus-free cells (cytoplasts), a 6 µg/cm2 dose of crocidolite increased oxyradical-related fluorescence by more than four-fold above control levels (71.1 vs 16.3 arbitrary units) at 20 minutes.
  • In crocidolite-treated cytoplasts, concurrent 0.5% DMSO reduced the fluorescence by more than two-fold.
  • Cytoplasts were treated with crocidolite (4 µg/cm2 for 3.5 h) and then fused with nuclei from untreated cells. In these reconstituted cells, the mutation yield was more than two-fold that of control fused cultures.
  • In the fused cells, concurrent 0.5% DMSO reduced the mutation yield by five-fold, to 43 plus or minus 17 per 10^5 survivors.
  • In whole AL cells exposed to crocidolite at 4 µg/cm2, L-NMMA at 15 µM suppressed the mutation yield by more than three-fold.
  • The inactive form D-NMMA had essentially no effect on the yield of mutants in crocidolite-exposed AL cells.
  • In AL cells, crocidolite produced a dose-dependent increase in nitric oxide production after 6 hours of treatment, peaking at 6 µg/cm2.
  • In AL cells treated with crocidolite at 6 µg/cm2, L-NMMA at 15 µM significantly suppressed nitric oxide formation by 5-6-fold.
  • In whole cells, a 6 µg/cm2 dose of crocidolite raised the peroxynitrite-related fluorescence to about three-fold higher than background.
  • In cytoplasts treated with crocidolite at 2 µg/cm2, the average peroxynitrite-related fluorescence was 2.4-fold above that of untreated cytoplasts.
  • Crocidolite caused a dose-dependent increase in nitrated proteins. Adding L-NMMA to the crocidolite-treated group (6 µg/cm2) reduced protein modification by 37%.
• The authors' conclusion: The authors concluded that targets outside the nucleus (extranuclear targets) play an essential role in starting the oxidative damage involved in fiber-induced mutations in mammalian cells.
• Limitations the authors state:
  • Because crocidolite fibers persist, treated cytoplasts were likely to contain some fiber remnants even after washing. The interaction between fibers and cytoplasm was therefore unlikely to have been completely eliminated after fusion with untreated nuclei.
  • The spontaneous (background) mutant level in the fused cells was about two-fold higher than normal for AL cells, possibly because of enhanced oxidative stress in enucleated cultures.
  • Only 15-20% of cells successfully fused.

Topics

The National Library of Medicine indexes this study under subjects our site covers: Crocidolite.

Medical terms in this study, explained in our Lexicon: Carcinogenic, Reactive Oxygen Species (ROS), Reactive Nitrogen Species (RNS), Crocidolite.

Related studies

• Effects of antioxidants on fiber mutagenesis — T.K. Hei, Z.Y. He, et al. (1995)
• Loss of heterozygosity in asbestos-induced mutations in a human mesothelioma cell line — K. Both, D.R. Turner, et al. (1995)
• Mechanisms of the genotoxicity of crocidolite asbestos in mammalian cells: implication from mutation patterns induced by reactive oxygen species — A. Xu, H. Zhou, et al. (2002)
• Modulation of genotoxic effects in asbestos-exposed primary human mesothelial cells by radical scavengers, metal chelators and a glutathione precursor — I. Poser, Q. Rahman, et al. (2004)
• The utility of the in vitro micronucleus test for evaluating the genotoxicity of natural and manmade nano-scale fibres — P. Fowler, A. Homan, et al. (2016)
• Reactive oxygen species, chromosome mutation, and cancer: possible role of clastogenic factors in carcinogenesis — I. Emerit (1994)

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